Method for gene editing

ABSTRACT

The present disclosure relates to compositions and methods for modifying a gene sequence, and for systems for delivering such compositions. For example, the disclosure relates to modifying a gene sequence using a CRISPR-Cas9 or other nucleic acid editing system, and methods and delivery systems for achieving such gene modification, such as viral or non-viral delivery systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/374,227, filed Dec. 9, 2016, which claims a priority benefit to PCTApplication No. PCT/US2015/035077, filed Jun. 10, 2015, entitled “METHODFOR GENE EDITING,” which is hereby incorporated by reference in itsentirety including drawings. PCT Application No. PCT/US2015/035077 inturn claims priority to U.S. Provisional Application Nos. 62/010,306,filed Jun. 10, 2014, entitled “METHODS FOR HIGH EFFICIENCY IN VIVO GENEEDITING”; 62/113,887, filed Feb. 9, 2015, entitled “METHOD FOR GENEEDITING”; and 62/156,562, filed May 4, 2015, entitled “METHOD FOR GENEEDITING”, each of which is incorporated herein by reference in itsentirety including drawings.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos. P01CA042063, P30 CA014051, and U54 CA151884 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

STATEMENT REGARDING SEQUENCE LISTING

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: MITX_7117_03US_ST25.txt,date recorded: Dec. 9, 2016, file size: 1.75 kb)

BACKGROUND OF THE INVENTION

The delivery of gene editing tools is a key challenge for therapeuticapplication of such tools. There is a need in the art for safe andeffective means of delivering gene editing tools to cells such that geneediting can be safely and effectively conducted in vivo or ex vivo.

SUMMARY OF THE INVENTION

The present disclosure provides delivery systems, compositions, methods,and kits for modifying a target nucleotide sequence in a subject. Thedisclosure provides for efficient in vivo gene editing and cellular DNAmodification using nucleic acid editing systems such as CRISPR-Cas9,among others.

In one aspect, the present disclosure provides delivery systemscomprising one or more guide RNA (gRNA) and a nucleic acid editingsystem. In one embodiment, the one or more gRNA is provided in a firstdelivery vehicle and the nucleic acid editing system is provided in asecond delivery vehicle. In one embodiment, the delivery system furthercomprises a repair template, wherein the repair template is provided inthe first, the second, or a third delivery vehicle.

In another aspect, the present disclosure provides delivery systemscomprising (i) one or more gRNA covalently or noncovalently bound to arepair template and (ii) a nucleic acid editing system, wherein (i) and(ii) are present on the same or different delivery vehicles.

In another aspect, the present disclosure provides methods, kits, andcompositions for modifying a target nucleotide sequence in a cell,comprising administering to the cell a delivery system comprising one ormore gRNA and a nucleic acid editing system. In one embodiment, thedelivery system comprises a first and second delivery vehicle asprovided herein, wherein the first and second delivery vehicles areadministered simultaneously or sequentially to the cell. In anotherembodiment, the one or more gRNA and/or the nucleic acid editing systemis administered to the cell in a plurality of administrations.

In one embodiment, the target sequence is present in a target cell, andthe target cell is present in a subject (i.e., in vivo). In anotherembodiment, the target cell has been isolated from a subject and ispresent ex vivo. In a further embodiment, the gRNA and nucleic acidediting system are administered to the cell ex vivo, and the ex vivomodified cell or cells may be re-introduced into the subject followingex vivo modification. In other embodiments, the target cell is in vitro.

In one embodiment, the subject is a mammal, such as a human, horse, cow,dog, cat, rodent, or pig. In particular embodiments, the subject is ahuman.

In one aspect, the present disclosure provides methods for treating adisease or disorder. In one embodiment, the disease or disorder is agenetic disease or disorder, a cancer, an inflammatory disease, or aninfection, such as an infection with a virus. In a further embodiment,the methods provided herein achieve a therapeutic effect in a subjectsuffering from a genetic disease or disorder, an inflammatory disease,or an infection. In one embodiment, the methods provided herein achievea target cell modification rate of about 0.01% to about 99%, or about0.1% to about 50%, or about 1% to about 10%.

In one embodiment, the present disclosure provides delivery systems,compositions, methods, and kits for modifying a target nucleotidesequence, comprising at least one delivery vehicle, wherein the at leastone delivery vehicle is a non-viral vector. In a further embodiment, thenon-viral vector is a lipid-based or polymeric vector. Lipid-based orpolymeric vectors may be selected, for example, from lipids, liposomes,lipid encapsulation systems, nanoparticles, small nucleic acid-lipidparticle (SNALP) formulations, polymers, and polymersomes. In oneembodiment, the polymer is selected from the group consisting of linearpolymers, branched polymers, dendrimers, and polysaccharides. In anotherembodiment, the lipid encapsulation system comprises one or more of aphospholipid, cholesterol, polyethylene glycol (PEG)-lipid, and alipophilic compound that delivers the particle to the target tissue. Ina further embodiment, the lipophilic compound is C12-200 or cKK-E12. Inone embodiment, the lipid encapsulation comprises1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterolC14-PEG2000, and cKK-E12.

In one embodiment, the non-viral vector is biodegradable. In anotherembodiment, the non-viral vector comprises at least one cell-targetingor tissue-targeting ligand. In one embodiment, the non-viral vector hasa size in the range of about 10 nm to about 10 μm, or about 20 nm toabout 5 μm, or about 50 nm to about 500 nm, or about 50 nm to about 200nm.

In one embodiment, the gRNA is delivered as an RNA conjugate. Thus, inone embodiment, the material conjugated to the gRNA acts as a non-viraldelivery vehicle. RNA conjugates, in one embodiment, include RNA-GalNAcconjugates and dynamic polyconjugates. In one embodiment, the gRNA ofthe RNA conjugate is chemically modified.

In one embodiment, at least one delivery vehicle is a viral vector.Viral vectors, in one embodiment, may be selected from adeno-associatedvirus (AAV), adenovirus, retrovirus, and lentivirus vectors. In oneembodiment, the viral vector is AAV 2/8.

In one aspect, the delivery system comprises a repair template. In oneembodiment, the repair template is a DNA repair template, an mRNA repairtemplate, an ssRNA repair template, an siRNA repair template, an miRNArepair template, or an antisense oligonucleotide repair template. In oneembodiment, the repair template is a DNA repair template. In oneembodiment, the length of the repair template is at least 200 bp, or isat least 500 bp, or is at least 800 bp, or is at least 1000 base pairs,or is at least 1500 base pairs. In one embodiment, the repair templateis covalently or noncovalently bound to the gRNA or to the nucleic acidediting system. In a further embodiment, the repair template ispartially annealed to the gRNA or to the nucleic acid editing system.

In one embodiment, the delivery system comprises a gRNA and a nucleicacid editing system, wherein the target sequence is recognized by thegRNA and modified by the nucleic acid editing system. In a furtherembodiment, the delivery system further comprises a repair template, andthe target sequence is modified by the nucleic acid editing system andrepair template. In one embodiment, the delivery system furthercomprises one or more reporter genes or epitope tags.

In one embodiment, the first delivery vehicle is a viral vector and thesecond delivery vehicle is a non-viral vector. In another embodiment,the first delivery vehicle is a non-viral vector and the second deliveryvehicle is a viral vector. In one embodiment, the first or the seconddelivery vehicle further comprises a repair template. In one embodiment,the first delivery vehicle is a viral vector comprising a gRNA and arepair template, and the second delivery vehicle is a non-viral vectorcomprising a nucleic acid editing system. In one embodiment, the gRNA isdelivered via an RNA conjugate, and the nucleic acid editing system isdelivered via a non-viral vector such as a nanoparticle. In anotherembodiment, the first and second delivery vehicles are both non-viralvectors, or are both viral vectors.

In one embodiment, the nucleic acid editing system is selected from thegroup consisting of ZFPs, TALEs, and CRISPR systems. In a furtherembodiment, the nucleic acid editing system is a CRISPR-Cas system suchas, for example, Cas9.

In one embodiment, the gRNA is expressed under the control of aninducible promoter. In another embodiment, the gRNA is expressed underthe control of a viral promoter. In another embodiment, the gRNA isexpressed under the control of a tissue-specific promoter. For example,in one embodiment, the promoter induces expression in one or more ofliver, heart, lung, skeletal muscle, CNS, endothelial cells, stem cell,blood cell or blood cell precursor, and immune cells. Promoters may beselected from the group consisting of U6, CMV, SV40, EF-1α, Ubc, PGK, orsmall molecule-inducible promoters, or other promoters known in the art.

In one embodiment, the gRNA and/or the RNA encoding the nucleic acidediting system is chemically modified. Chemical modifications of RNA mayinclude modifications of the phosphate backbone (e.g., phosphorothioatelinkages or boranophosphate linkages), ribose ring modifications such as2′-O-methyl and/or 2′-fluoro and/or 4′-thio modifications, and locked orunlocked nucleic acids. Other modifications may include pseudouridine,2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine,5-aminouridine, 5-methyluridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-aminopseudouridine, pseudoisocytidine, 5-methylcytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine,5-hydroxypseudoiocytidine, 5-aminopseudoisocytidine,5-methylpseudoisocytidine, N6-methyladenosine, 7-deazaadenosine,6-thioguanosine, 7-deazaguanosine, 8-azaguanosine,6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine,and 6-thio-7-deaza-8-azaguanosine.

In one embodiment, the gRNA is capable of hybridizing to a targetsequence in a cell. In a further embodiment, the target sequence islocated in the nucleus or cytoplasm of the cell.

In one embodiment, the target sequence is associated with a geneticdisease or disorder. Genetic diseases and disorders may be inborn errorsof metabolism selected from disorders of amino acid transport andmetabolism, lipid or fatty acid transport and metabolism, carbohydratetransport and metabolism, and metal transport and metabolism. In oneembodiment, the genetic disease or disorder is associated with a geneticvariant selected from a single-nucleotide polymorphism (SNP),substitution, insertion, deletion, transition, transversion,translocation, nonsense, missense, and frameshift mutation. In oneembodiment, the genetic disorder is hemophilia, cystic fibrosis, orsickle cell disease. In another embodiment, the target sequence is avirus or a provirus sequence. For example, in one embodiment, the targetsequence is a human immunodeficiency virus (HIV) or humanT-lymphotrophic virus (HTLV) sequence. In one embodiment, the targetsequence is associated with cancer. In a further embodiment, the targetsequence is a tumor driver gene. In one embodiment, the target sequenceis a gene associated with immune suppression in cancer.

In one aspect, the present disclosure provides kits and compositions,wherein the kits and compositions comprise (i) one or more gRNA in afirst delivery vehicle and (ii) a nucleic acid editing system in asecond delivery vehicle. In one embodiment, the kits and compositionsfurther comprise a repair template. In a further embodiment, the repairtemplate is covalently or noncovalently bound to the gRNA.

In one aspect, the present disclosure provides methods for modifying atarget nucleotide sequence in a cell or a subject, comprisingadministering to the cell or subject a delivery system, wherein thedelivery system provides for temporally controlled expression of a gRNAin a target tissue as well as temporally controlled expression of anucleic acid editing system in the target tissue, wherein the gRNAdirects cleavage of the target nucleic acid sequence in the targettissue by the nucleic acid editing system. In one embodiment, thedelivery system provides a gRNA and a nucleic acid editing system, andfurther provides a DNA repair template, wherein the gRNA directscleavage of the target nucleic acid sequence in the target tissue andrepair of the target sequence by the repair template.

In one embodiment, the gRNA is expressed in the cell prior to thenucleic acid editing system. In another embodiment, the nucleic acidediting system is expressed transiently in the cell. In one embodiment,the gRNA is delivered to the target cell or tissue in an AAV vector andthe nucleic acid editing system is delivered to the target cell ortissue in a lipid-based delivery vehicle, wherein the gRNA is deliveredto the cell or tissue prior to the nucleic acid editing system. In afurther embodiment, the gRNA is delivered to the cell or tissue about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days prior to thedelivery of the nucleic acid editing system. In another embodiment, thegRNA and/or the nucleic acid editing system is administered to thetarget cell or tissue in a plurality of administrations. For example, inone embodiment, the nucleic acid editing system is delivered to thetarget cell or tissue in from about 2 to about 20 administrations. Inone embodiment, the nucleic acid editing system is administered in alipid-based delivery vehicle about 7 days and about 14 days after theadministration of the one or more gRNA in an AAV vector. In oneembodiment, the nucleic acid editing system is delivered to the targetcell or tissue over a time period of from about 1 week to about 6months, such as from about 2 to about 10 doses within about 2 months, orfrom about 3 to about 5 doses over about 1 month.

In one embodiment, the gRNA is expressed in the target cell or tissuefor about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks,9 weeks, 10 weeks, 11 weeks, 12 weeks, or more. In another embodiment,the nucleic acid editing system is expressed in the target cell ortissue transiently. In a further embodiment, the nucleic acid editingsystem is expressed in the target cell or tissue for less than a month,less than 3 weeks, less than 2 weeks, less than 1 week, less than 5days, or less than 3 days. For example, in one embodiment, the nucleicacid editing system is expressed in the target tissue for about 1 day toabout 5 days, or for about 1 day to about 3 days. Thus, the vehicleproviding for expression of the gRNA(s) and optional presence of the DNArepair template provides a window of time in which a nucleic acidediting system can be administered (once or a plurality of times) toprovide transient expression of the editing system (e.g., nuclease), aswell as a level of tissue-specific expression in some embodiments,thereby directing editing of the cellular DNA.

The delivery systems and compositions disclosed herein may beadministered by injection, optionally by direct injection to targettissues.

In one embodiment, the delivery systems and compositions disclosedherein may be administered to a subject in more than one dose. In oneembodiment, the nucleic acid editing system is administered to thesubject in a non-viral delivery vehicle (e.g., a lipid-based deliveryvehicle) in more than one dose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the repair of a Fumarylacetoacetatehydrolase (FAH) mutation, which is a mouse model for the human diseasetyrosinemia I. The sgRNA targeting the region of the FAH mutation isexpressed through a U6 promoter in the AAV 2/8 vector. A repair templateis also provided in the vector.

FIG. 2A is a schematic depiction of the Cas9 mRNA packing into the lipidnanoparticle cKK-E12. FIG. 2B is a Western blot showing expression ofCas9 mRNA (containing a human influenza hemagglutinin (HA) tag) in livertissue from FVB/N mice injected with the lipid nanoparticle comprisingthe Cas9 mRNA.

FIGS. 3A (low resolution) and 3B (high resolution) show FAH expressionat Day 21 in the liver of FAH mutant mice that had received the AAV 2/8delivery vehicle on Day 0, and at Day 7 and Day 14 received 1 mg/kg ofthe lipid nanoparticle encapsulated Cas9 mRNA (or PBS as a control). Theleft panels of FIGS. 3A and 3B show FAH expression from mice thatreceived the AAV 2/8 delivery vehicle only. The right panels of FIG. 3Aand 3B show FAH expression in mice that received the AAV 2/8 deliveryvehicle and the lipid nanoparticle delivery vehicle.

FIG. 4 is a set of histographs showing EGFP expression in untreatedHEK293T cells (left panel), HEK293T cells treated with MD-1 expressingCas9 mRNA (center panel), or C112-200 expressing Cas9 mRNA (rightpanel).

FIG. 5 provides the selection of the most potent sgRNA in vitro. sgRNAswere screened in a cell line established from an FAH mouse. A surveyorassay was performed to determine the efficiency of indels formation.

FIG. 6 provides selection of the most potent sgRNA in vivo. sgRNA werescreened in the liver through hydrodynamic injection, and a surveyorassay was performed to determine the efficiency of indels formation.

FIG. 7 provides the in vivo and in vitro correlations of indelsformation by deep sequencing.

FIGS. 8A-8E show that in vitro delivery data of Cas9 mRNA mediatesefficient genome editing in cells. FIG. 8A: C12-200 lipid nanoparticledelivery of Cas9 mRNA into cells. 293T cells stably expressing EF1apromoter-GFP and U6 promoter-GFP targeting sgRNA (sgGFP) were incubatedwith Cas9 mRNA nanoparticles (nano.Cas9). Cas9-mediated frameshift NHEJevents will result in GFP-negative cells. Red arrowhead indicates theCas9 cutting site. FIG. 8B: FACS analysis shows that Cas9 mRNA generatesGFP-cells. Gate R2 indicates 80% GFP-cells after nano.Cas9 treatment.FIG. 8C: GFP locus was deep sequenced in nano.Cas9 treated cells (n=2).Shown are representative indels. FIG. 8D: Distribution of indels. FIG.8E: Indel phase shows that most indels cause frameshift. For example,3N+1 include 1-, 4- and 7-bp indels, 3N+2 include 2-, 5- and 8-base-pairindels and 3-, 6- and 9-base-pair indels are 3N.

FIGS. 9A-9E provide in vivo delivery of Cas9 mRNA and AAV-sgRNA-HDRtemplate cures type I tyrosinemia mice. FIG. 9A: Design of dual functionAAV-sgRNA-HDR template (AAV-HDR). A G->A point mutation at the lastnucleotide of exon 8 in Fah^(mut/mut) homozygous mice leads to splicingskipping of exon 8. A dual function AAV vector harbors U6-sgRNA and aHDR template (1.7kb) with the “G” nucleotide to repair the “A” mutation.The “TGG” PAM was modified to “TCC” to preventing self-cleavage. Dashedlines denote homozygous recombination. ITR, inverted terminal repeat.FIG. 9B: Fah^(mut/mut) mice were injected with AAV-HDR and nano.Cas9 atindicated time points. Mice were kept off NTBC water at D0. Body weightnormalized to pre-injection was monitored over time. FIG. 9C: AAV-HDRand nano.Cas9 fully rescues weight loss upon NTBC withdrawal. FIG. 9D:Liver damage markers (aspartate aminotransferase (AST), alanineaminotransferase (ALT) and bilirubin) were measured in serum. Errorbars, mean±s.e.m. FIG. 9E: FAH+ cells after 30 Days off NTBC. Arrow Barindicates 100 uM

FIGS. 10A-10F provide in vivo delivery of Cas9 mRNA and AAV corrects Fahmutation. FIG. 10A: Fah^(mut/mut) mice were kept on NTBC water andeuthanized 7 days after nano.Cas9 treatment to estimate initial repairrate. FIG. 10B: Fah immunohistochemistry (IHC). FIG. 10C: The percentageof FAH+ positive cells were counted. FIG. 10D: Quantitative RT-PCRmeasurement of wild-type the expression of Fah mRNA. FIG. 10E: Sequenceof repaired Fah mRNA in treated mice. The corrected G nucleotide iscircled. FIG. 10F: indels of total DNA from liver.

FIGS. 11A-11E provide Cas9 mRNA nanoparticles characterization. FIG.11A: nano.Cas9 formulation scheme. Cas9 mRNA was mixed with C12-200,DOPE, Cholesterol and C14PEG2000 in a microfluidic chamber. FIG. 11B:nano.Cas9 structure is characterized by cryo-TEM. Scale bar indicates100nm. FIG. 11C: Average diameter of nano.Cas9 was measured by dynamiclight scattering. The size of nano.Cas9 FIG. 11D and the polydispersityindex (PDI) FIG. 11E were measured 0, 7, 11 or 18 days after formulationand storage at 4° C.

FIGS. 12A-12E provide data on expression of proteins in mouse liverafter mRNA nanoparticles treatment. FIG. 12A: C57bl/6 mice were i.v.injected with nanoparticles encapsulated with β-gal (B and C) or Cas9mRNA (nano.Cas9, D and E), and livers taken. FIG. 12B: The expression ofβ-gal protein is measured in liver lysate at 14 hours after injection.FIG. 12C: The activity of β-gal in liver sections was determined bysalmon-gal assay. Scale bar indicates 200 μm. FIG. 12D: The Cas9 mRNAlevel in liver lysate was determined by qRT-PCR at 4, 14, and 24 hrsafter injection. FIG. 12E: The expression of Cas9 protein was measuredin liver lysate 14 hours after injection. 10, 1 or 0.1 ng Cas9 proteinmixed into 50 μg negative control samples were served as positivecontrols.

FIGS. 13A-13D provide results demonstrating that Cas9 mRNA nanoparticlesare well tolerated. FIG. 13A: 293T cells were transfected with Cas9 mRNAwith Lipo2000 or nano.Cas9, and cellular viability was determined 48 hrslater (B-D). C57/B16 mice were treated with 2 mg/kg nano.Cas9, andhistology FIG. 13B, liver damage enzymes FIG. 13C and plasma cytokinesFIG. 13D were determined after 24 hrs. Scale bar indicates 50 μm.

DETAILED DESCRIPTION

The term “about”, as used herein, refers to plus or minus ten percent ofthe object that “about” modifies.

The present disclosure provides delivery systems, compositions, methods,and kits for modifying a target nucleotide sequence in a cell. In someembodiments, the delivery systems comprise one or more guide RNA (gRNA)and a nucleic acid editing system. The gRNA works in tandem with thenucleic acid editing system to localize to and edit the target cellularnucleotide sequence. The delivery systems, compositions, methods, andkits provided herein allow for temporally controlled expression of thegRNA and the nucleic acid editing system. Optionally, a repair templatemay be included to replace the target nucleotide sequence thus effectingeither repair of a gene defect or knock-in of a selected sequence. Inparticular aspects, the nucleic acid editing system is a CRISPR-Cas9system. The CRISPR (clustered regularly interspersed short palindromicrepeats)/Cas9 system has emerged as a genetic editing tool, as disclosedin, for example Cong et al. Multiplex genome engineering usingCRISPR/Cas systems. Science. 339:819-823 (2013); Doudna and Charpentier.Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 346:1258096 (2014); Mali et al. RNA-guided human genomeengineering via Cas9. Science. 339:823-826. (2013). CRISPR-Cas9 has beenemployed to edit genomes of various model organisms such as bacteria,yeast, C. elegans, Drosophila, plants, zebrafish, and mouse and humancells, as disclosed in, for example, Mali et al. Cas9 as a versatiletool for engineering biology, Nat. Methods. 10:957-963 (2013).

Cas9/sgRNA recognizes the protospacer-adjacent motif (PAM) sequence andthe complementary 20 nucleotide genomic sequence. Cas9 cutsapproximately 3 nucleotides upstream of the PAM to induce doublestranded DNA breaks (DSBs), which are repaired by error-pronenon-homologous end-joining (NHEJ) or precise homology-directed repair(HDR), as disclosed in, for example, Doudna and Charpentier (2014); andSander and Joung. CRISPR-Cas systems for editing, regulating andtargeting genomes. Nat. Biotechnol. 32:347-355 (2014). Improvements toCRISPR delivery methods and HDR efficiency are needed in the field fortherapeutic application of genome editing for disease gene correction.

Nucleic Acid Editing Systems

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA nuclease enzymeassociated with Streptococcus pyogenes CRISPR immunity system. Cas9 canbe used to induce site-directed double strand breaks in DNA, which canlead to gene inactivation or the introduction of heterologous genesthrough non-homologous end joining and homologous recombinationrespectively. mRNA systems for expressing Cas9 are commerciallyavailable from TriLink Biotechnologies (San Diego, Calif.). The mRNA maybe codon optimized for human or other mammalian system. The expressedCas9 protein may contain a nuclear localization signal at theC-terminus. The RNA encoding Cas9 may be capped and polyadenylated tosupport expression in mammalian cells, and may contain modifications toreduce immune stimulation. The amino acid sequence and encoding nucleicacid sequence for Cas9 and functional derivatives and homologs (whichcan be used in accordance with the disclosure) include those describedin U.S. Pat. No. 8,697,359, which is hereby incorporated by reference inits entirety.

The Cas9 may be delivered in conjunction with a gRNA, which directs theCas9 editing system to the nucleotide sequence recognized by the gRNA.The term “gRNA” is used interchangeably herein with “gRNA” “singlegRNA,” and “sgRNA.” In general, a gRNA can be designed to target anynucleotide sequence. The gRNA structure is disclosed in, for example,Ran F A, Genome editing using the CRISPR-Cas9 System, PNAS8(11):2281-308 (2013); and Pyzocha et al., RNA-guided genome editing ofmammalian cells, Methods Mol. Biol. 1114:269-77 (2014), which are herebyincorporated by reference in their entirety. Generally for Cas9, gRNAsguide the Cas9 endonuclease to the complementary 20 nucleotide (nt)genomic sequences with a downstream NGG protospacer-adjacent motif(PAM). Cas9 generates double-stranded breaks, which can be repaired bynon-homologous end-joining (NHEJ) or homologous recombination (HR). See,for example, US 2014/0017212, which is hereby incorporated by referencein its entirety.

The CRISPR-Cas9 system including the construction of guide sequences isfurther disclosed in U.S. Pat. No. 8,697,359, which is herebyincorporated by reference in its entirety. In some embodiments, a Cas9nickase version is employed. Cas9 nickase can generate single strandedbreaks, and a double nickase can generate double stranded breaks. Thenickase can provide for reduced off-target effects. Further, bydelivering two gRNAs and a Cas9 nickase, off target effects can befurther reduced. See Shen et al., Efficient genome modification byCRISPR-Cas9 nickase with minimal off-target effects, Nature Methods11:399-402 (2014).

In place of a CRISPR-Cas9 system, alternate nucleic acid editing systemsmay be used. For example, suitable systems include any CRISPR/cas system(e.g., any Cascade-like CRISPR/cas, Type I CRISPR/cas, Type IICRISPR/cas, and type III CRISPR/cas), zinc finger nucleases (ZFNs),transcription activator-like effector nucleases (TALENs), and engineeredmeganuclease re-engineered homing endonucleases.

RNA encoding the nucleic acid editing system can be modified, and themodification selected from one or more of modifications of the phosphatebackbone (e.g., phosphorothioate linkages or boranophosphate linkages),ribose ring modifications such as 2′-O-methyl and/or 2′-fluoro and/or4′-thio modifications, and locked or unlocked nucleic acids. Othermodifications may include pseudouridine, 2-thiouridine, 4-thiouridine,5-azauridine, 5-hydroxyuridine, 5-aminouridine, 5-methyluridine,2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine,5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine,5-methylcytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine,5-hydroxycytidine, 5-aminocytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, 5-methylpseudoisocytidine, N6-methyladenosine,7-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine,6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine,and 6-thio-7-deaza-8-azaguanosine. Generally, modifications are selectedto reduce immune stimulation and stabilize the RNA and improveexpression of the encoded protein. For example, the RNA may have acombination of 2-thiouridine and 5-methyl-cytidine, which has been shownto reduce immune stimulation through pattern recognition receptors, suchas TLR3, TLR7, TLR8 and RIG-I (retinoic-acid-inducible protein I). Insome embodiments, the mRNA has one or more pseudouridine (preventingactivation of pattern recognition receptors and 2′-5′-oligoadenylatesynthetase). These modifications can also stabilize the mRNA againstcleavage, and ultimately improve expression rates.

Delivery Systems

The efficient delivery of nucleic acid editing systems, including theCRISPR-Cas9 system, provide for safer and more effective deliverysystems, which are especially useful in the clinical setting. Thedelivery systems herein disclose methods and compositions containingviral and/or non-viral vectors to deliver nucleic acid editing systems,particularly, CRISPR-Cas9 system, and optionally an editing template toedit genes in cells. While gene editing is particularly useful in vivo,in some embodiments, the cell targeted for gene editing may be in vitro,ex vivo, or in vivo.

Delivery Vehicles

The delivery vehicles provided herein may be viral vectors or non-viralvectors, or RNA conjugates. In some embodiments, the gRNA and thenucleic acid editing system are provided in the same type of deliveryvehicle, wherein the delivery vehicle is a viral vector or a non-viralvector. In other embodiments, the gRNA is provided in a viral vector,and the nucleic acid editing system is provided in a non-viral vector.In still other embodiments, the one or more gRNA is provided in anon-viral vector and the nucleic acid editing system is provided in aviral vector. In some embodiments, the gRNA is provided in an RNAconjugate.

Viral Vectors

In some embodiments, the viral vector is selected from anadeno-associated virus (AAV), adenovirus, retrovirus, and lentivirusvector. While the viral vector may deliver any component of the systemdescribed herein so long as it provides the desired profile for tissuepresence or expression, in some embodiments the viral vector providesfor expression of the gRNA and optionally delivers a repair template. Insome embodiments, the viral delivery system is adeno-associated virus(AAV) 2/8. However, in various embodiments other AAV serotypes are used,such as AAV1, AAV2, AAV4, AAV5, AAV6, and AAV8. In some embodiments,AAV6 is used when targeting airway epithelial cells, AAV7 is used whentargeting skeletal muscle cells (similarly for AAV1 and AAV5), and AAV8is used for hepatocytes. In some embodiments, AAV1 and 5 can be used fordelivery to vascular endothelial cells. Further, most AAV serotypes showneuronal tropism, while AAV5 also transduces astrocytes. In someembodiments, hybrid AAV vectors are employed. In some embodiments, eachserotype is administered only once to avoid immunogenicity. Thus,subsequent administrations employ different AAV serotypes. Additionalviral vectors that can be employed are as described in U.S. Pat. No.8,697,359, which is hereby incorporated by reference in its entirety.

Non-Viral Vectors

In some embodiments, the delivery system comprises a non-viral deliveryvehicle. In some aspects, the non-viral delivery vehicle is lipid-based.In other aspects, the non-viral delivery vehicle is a polymer. In someembodiments, the non-viral delivery vehicle is biodegradable. Inembodiments, the non-viral delivery vehicle is a lipid encapsulationsystem and/or polymeric particle.

Lipid-Based and Polymeric Non-Viral Vectors

In certain embodiments, the delivery system comprises lipid particles asdescribed in Kanasty R, Delivery materials for siRNA therapeutics NatMater. 12(11):967-77 (2013), which is hereby incorporated by reference.In some embodiments, the lipid-based vector is a lipid nanoparticle,which is a lipid particle between about 1 and about 100 nanometers insize.

In some embodiments, the lipid-based vector is a lipid or liposome.Liposomes are artificial spherical vesicles comprising a lipid bilayer.

In some embodiments, the lipid-based vector is a small nucleicacid-lipid particle (SNALP). SNALPs comprise small (less than 200 nm indiameter) lipid-based nanoparticles that encapsulate a nucleic acid. Insome embodiments, the SNALP is useful for delivery of an RNA moleculesuch as siRNA. In some embodiments, SNALP formulations deliver nucleicacids to a particular tissue in a subject, such as the liver.

In some embodiments, the gRNA and/or nucleic acid editing system (or theRNA encoding the same) is delivered via polymeric vectors. In someembodiments, the polymeric vector is a polymer or polymerosome. Polymersencompass any long repeating chain of monomers and include, for example,linear polymers, branched polymers, dendrimers, and polysaccharides.Linear polymers comprise a single line of monomers, whereas branchedpolymers include side chains of monomers. Dendrimers are also branchedmolecules, which are arranged symmetrically around the core of themolecule. Polysaccharides are polymeric carbohydrate molecules, and aremade up of long monosaccharide units linked together. Polymersomes areartificial vesicles made up of synthetic amphiphilic copolymers thatform a vesicle membrane, and may have a hollow or aqueous core withinthe vesicle membrane.

Various polymer-based systems can be adapted as a vehicle foradministering RNA encoding the nucleic acid editing machinery. Exemplarypolymeric materials include poly(D,L-lactic acid-co-glycolic acid)(PLGA), poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA),poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid)(PGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), PLGA-b-poly(ethylene glycol)-PLGA(PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG-maleimide (PLGA-PEG-mal),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)(polyacrylic acids), and copolymers and mixtures thereof, polydioxanoneand its copolymers, polyhydroxyalkanoates, polypropylene fumarate),polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone), trimethylenecarbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes,Poly([beta]-amino esters (PBAE), and polyphosphoesters, and blendsand/or block copolymers of two or more such polymers. Polymer-basedsystems may also include Cyclodextrin polymer (CDP)-based nanoparticlessuch as, for example, CDP-admantane (AD)-PEG conjugates andCDP-AD-PEG-transferrin conjugates.

Exemplary polymeric particle systems for delivery of drugs, includingnucleic acids, include those described in U.S. Pat. Nos. 5,543,158,6,007,845, 6,254,890, 6,998,115, 7,727,969, 7,427,394, 8,323,698,8,071,082, 8,105,652, US 2008/0268063, US 2009/0298710, US 2010/0303723,US 2011/0027172, US 2011/0065807, US 2012/0156135, US 2014/0093575, WO2013/090861, each of which are hereby incorporated by reference in itsentirety.

In one embodiment, nanoparticles are formulated with Cas9 mRNAchemically modified to reduce TLR responses, as disclosed in Kormann etal. Expression of therapeutic proteins after delivery of chemicallymodified mRNA in mice. Nat. Biotechnol. 29:154-157 (2011). In a furtherembodiment, the nanoparticles are formulated using controlledmicrofluidic mixing systems, as disclosed in, for example, Chen et al.Rapid discovery of potent siRNA-containing lipid nanoparticles enabledby controlled microfluidic formulation. J. Amer. Chem. Soc.134:6948-6951 (2012).

In one embodiment, the delivery system is a layer-by-layer particlesystem comprising two or more layers. In a further embodiment, the guideRNA and the nucleic acid editing system are present in different layerswithin the layer-by-layer particle. In a yet further embodiment, theguide RNA and nucleic acid editing system may be administered to asubject in a layer-by-layer particle system such that the release of theguide RNA and nucleic acid editing system from the particles can becontrolled in a cell-specific and/or temporal fashion. In oneembodiment, the layer-by-layer particle system is designed to allowtemporally controlled expression of the guide RNA and the nucleic acidediting system as disclosed herein. Layer-by-layer particle systems aredisclosed, for example, in 2014/0093575, incorporated herein byreference in its entirety.

Lipid Encapsulation System Vectors

In some embodiments, the lipid-based delivery system comprises a lipidencapsulation system. The lipid encapsulation system can be designed todrive the desired tissue distribution and cellular entry properties, aswell as to provide the requisite circulation time and biodegradingcharacter. The lipid encapsulation may involve reverse micelles and/orfurther comprise polymeric matrices, for example as described in U.S.Pat. No. 8,193,334, which is hereby incorporated by reference. In someembodiments, the particle includes a lipophilic delivery compound toenhance delivery of the particle to tissues, including in a preferentialmanner. Such compounds are disclosed in US 2013/0158021, which is herebyincorporated by reference in its entirety. Such compounds may generallyinclude lipophilic groups and conjugated amino acids or peptides,including linear or cyclic peptides, and including isomers thereof. Anexemplary compound is referred to as cKK-E12, which can affect deliveryto liver and kidney cells, for example. The present disclosure canemploy compounds of formulas (I), (II), (III), IV), (V), and (VI) of US2013/0158021. Compounds can be engineered for targeting to varioustissues, including pancreas, spleen, liver, fat, kidneys,uterus/ovaries, muscle, heart, lungs, endothelial tissue, and thymus.

In some embodiments, the lipid encapsulation comprises one or more of aphospholipid, cholesterol, polyethylene glycol (PEG)-lipid, and alipophilic compound. In some embodiments, the lipophilic compound isC12-200, particularly in embodiments that target the liver. (Love etal., Lipid-like materials for low-dose, in vivo gene silencing PNAS107(21) (2010), incorporated herein by reference in its entirety. Inother embodiments, the lipophilic compound C12-200 is useful inembodiments that target fat tissue. In still other embodiments, thelipopeptide is cKK-E12. Dong, et al., PNAS. 111(11):3955-3960 (2014),incorporated herein by reference in its entirety.

In some embodiments, the lipid encapsulation comprises1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol,C14-PEG2000, and cKK-E12, which as disclosed herein provides forefficient in vivo editing in liver tissue. An illustration of such aparticle is shown in FIG. 2A-B.

Additional Components and Features of Non-Viral Vectors

The particles, whether lipid or polymeric or both, may includeadditional components useful for enhancing the properties for in vivonucleic acid delivery (including compounds disclosed in U.S. Pat. No.8,450,298 and US 2012/0251560, which are each hereby incorporated byreference).

The delivery vehicle may accumulate preferentially in certain tissuesthereby providing a tissue targeting effect, but in some embodiments,the delivery vehicle further comprises at least one cell-targeting ortissue-targeting ligand. Functionalized particles, including exemplarytargeting ligands, are disclosed in US 2010/0303723 and 2012/0156135,which are hereby incorporated by reference in their entireties.

A delivery vehicle can be designed to drive the desired tissuedistribution and cellular entry properties of the delivery systemsdisclosed herein, as well as to provide the requisite circulation timeand biodegrading character. For example, lipid particles can employamino lipids as disclosed US 2011/0009641, which is hereby incorporatedby reference.

The lipid or polymeric particles may have a size (e.g., an average size)in the range of about 50 nm to about 5 μm. In some embodiments, theparticles are in the range of about 10 nm to about 100 μm, or about 20nm to about 50 μm, or about 50 nm to about 5 μm, or about 70 nm to about500 nm, or about 70 nm to about 200 nm, or about 50 nm to about 100 nm.Particles may be selected so as to avoid rapid clearance by the immunesystem. Particles may be spherical, or non-spherical in certainembodiments.

In some embodiments, the non-viral delivery vehicle may be a peptide,such as a cell-penetrating peptides or cellular internalizationsequences. Cell penetrating peptides are small peptides that are capableof translocating across plasma membranes. Exemplary cell-penetratingpeptides include, but are not limited to, Antennapedia sequences, TAT,HIV-Tat, Penetratin, Antp-3A (Ante mutant), Buforin II, Transportan, MAP(model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1,Pep-7, I-IN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC(Bis-Guanidinium-Tren-Cholesterol).

Chemical Modification of RNA and RNA Conjugates

In some embodiments, the gRNA is chemically modified. In otherembodiments, the gRNA is conjugated to a material to aid in delivery ofthe RNA to the target tissue or cell. Thus, in some embodiments, thematerial to which the gRNA is conjugated acts as a gRNA deliveryvehicle. In further embodiments, the gRNA of the RNA conjugate ischemically modified. Any chemical modification or conjugate material maybe used in the delivery systems, methods, and compositions providedherein, including those chemical modifications and conjugate materialsdisclosed in Kanasty et al., Nature Materials 12; 967 (2013).

Chemical modification of the RNA molecule may stabilize the moleculeprior to reaching the target cell (e.g., in the bloodstream), reduceimmunogenicity of the RNA molecule, and improve delivery to the targetcell and/or improve entry into the target cell. Chemical modificationsof RNAs are known in the art, for example, in Kanasty et al., NatureMaterials 12; 967 (2013) and Corey, D R. Journal of ClinicalInvestigation 117; 3615 (2007), each of which is incorporated herein byreference in its entirety. Chemical modifications of RNA may includemodifications of the phosphate backbone (e.g., phosphorothioate linkagesor boranophosphate linkages), ribose ring modifications such as2′-O-methyl and/or 2′-fluoro and/or 4′-thio modifications, and locked orunlocked nucleic acids. Other modifications may include pseudouridine,2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine,5-aminouridine, 5-methyluridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-aminopseudouridine, pseudoisocytidine, 5-methylcytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine,5-hydroxypseudoisocytidine, 5-aminopseudoi socytidine, 5-methylpseudoisocytidine, N6-methyladenosine, 7-deazaadenosine, 6-thioguanosine,7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine,6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and6-thio-7-deaza-8-azaguanosine.

In some embodiments, the RNA molecule is modified at the 5′ end (e.g.,the 20-25 nucleotides at the amino terminus, the middle portion of theRNA (e.g., the Cas9 binding portion, which is about 42 nucleic acidslong), or the 3′ end (e.g., the 30-35 nucleic acids at carboxy terminusof the RNA). In a preferred embodiment, the modification is made at the3′ end of the RNA.

The RNA may be conjugated to cholesterol, other lipophilic molecules,polymers, peptides, antibodies, aptamers, and/or small molecules. Insome embodiments, the RNA is conjugated to a N-acetylgalactosamine(GalNAc). GalNAc binds the asialoglycoprotein receptor (ASGPR) onhepatocytes, and therefore can be used to target an RNA to the liver. Insome embodiments, the RNA is conjugated to a trivalent targeting ligand,e.g., triantennary GalNAc. Such conjugates comprise an RNA conjugated atthe 3′ terminus to three GalNAc molecules. Exemplary RNA-GalNAcconjugates are disclosed, for example, in Kanasty et al., NatureMaterials 12; 967 (2013), incorporated herein by reference in itsentirety.

In one embodiment, the conjugate delivery system is a dynamicpolyconjugate (DPC) system. In one embodiment, a DPC comprises amembrane-disrupting polymer linked to the RNA molecule via linker suchas a hydrolysable disulphide linker. The membrane-disrupting polymer maybe poly(butyl amino vinyl ether; PBAVE). In a further embodiment, PEGside chains are linked to the polymer backbone. In one embodiment, thePEG side chains mask the polymer and induce uptake by a target cell (viareceptor-mediated endocytosis), after which PEG is shed in the endosome,exposing the membrane-disrupting polymer and triggering release from theendosome. After endosomal release, the linker is cleaved (e.g.,disuphide cleavage in the cytosol) and the RNA is released into thecell. In some embodiments, the membrane-disrupting polymer further isattached to a targeting ligand such as, for example, GalNAc. In oneembodiment, the RNA molecule in the DPC system is chemically modifiedaccording to the chemical modifications disclosed herein.

Duration of Expression

In some aspects, the delivery vehicle for the gRNA is selected such thatthe gRNA is expressed in the target tissue for at least 1 week. However,longer expression will be desired in some embodiments, such asexpression in the target tissue for at least 2 weeks, 3 weeks, or atleast 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7weeks, or at least 8 weeks, or for at least 2 months, at least 3 months,at least 4 months, at least 6 months, at least 8 months, at least 10months, at least 12 months, at least 18 months, at least 24 months, ormore. In some embodiments, the length of time of expression of the gRNAprovides a window in which an editing system is provided to the cells toeffect the nucleic acid modification.

In some embodiments, the delivery systems, compositions, methods, andkits provided herein provide transient expression of the nucleic acidediting system (e.g., Cas9) in the target cell. In some embodiments,such transient expression helps to minimize off-target effects and/orimmunogenicity. For example, in one embodiment, the delivery systems,compositions, methods, and kits provided herein provide expression ofthe nucleic acid editing system such as Cas9 in a cell for about twoweeks or less, or for about 1 week or less. In some embodiments, thecompositions and delivery systems provided herein provide expression ofthe nucleic acid editing system such as Cas9 in a cell for about 1 dayto about 5 days, or for about 1 day to about 3 days.

The timing and type of expression of the gRNA and/or nucleic acidediting system such as Cas9 can be varied, such as throughtissue-specific promoters, constitutive promoters or induciblepromoters. As used herein, an inducible promoter is any promoter whoseactivity is regulated upon the application of an agent, such as a smallmolecule, to the cell. For example, inducible promoters includetetracycline-regulatable or doxycycline-regulatable promoters,carbohydrate-inducible or galactose-inducible promoters,isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoters,heat-shock promoters, and steroid-regulated promoters. In certainembodiments, the nucleic acid editing system and/or gRNA is expressedfrom a tissue specific promoter, e.g., a promoter that is active in thetarget tissue more than some other tissues. For example, depending onthe target tissue, the promoter is a tissue specific promoter that isexpressed in one or more of liver, heart, lung, skeletal muscle, CNS,endothelial cells, stem cell, blood cell or blood cell precursor, andimmune cells. Exemplary promoters include RNA III polymerase promoters,and viral promoters such as U6, CMV, SV40, EF-1α, Ubc, and PGKpromoters, or derivatives thereof having comparable promoter strength.Other promoters can be selected and/or designed based on publiclyavailable information (see, for example, the mammalian promoter databaseat mpromdb.wistar.upenn.edu). These and other promoters, expressioncontrol elements (e.g., enhancers), and constructs that can be used aredescribed, for example, in U.S. Pat. No. 8,697,359, which is herebyincorporated by reference in its entirety.

The duration of expression of the nucleic acid editing system and/orgRNA can be determined in a suitable cell line that is indicative ofexpression in the target tissue, and/or where the promoter of choice isexpressed in a manner that is comparable with the target tissue. Forexample, where the target tissue is liver, the duration of expression ofthe nucleic acid editing system and/or gRNA may be determined inhepatocyte cell culture such as HuH-7 or transformed primary humanhepatocytes. Alternatively, Human Embryonic Kidney 293T cells may beused to quantify duration of expression. Expression can be measured by,for example, immunohistochemistry, RT-PCR, or flow cytometry. In someembodiments, a gRNA or nucleic acid editing system such as Cas9, forexample, can be expressed with a suitable tag (e.g., HA tag) to monitorexpression with commercially available antibodies. In some embodiments,the expression of the nucleic acid editing system and/or gRNA and/or theefficiency of target nucleotide modification can be detected ormonitored using reporter genes, reporter sequences, epitope tags, and/orexpression tags. A “reporter gene” or “reporter sequence” or “epitopetag” or “expression tag” refers to any sequence that produces a productthat is readily measured. Reporter genes include, for example, sequencesencoding proteins that mediate antibiotic resistance (e.g., ampicillinresistance, neomycin resistance, G418 resistance, puromycin resistance),sequences encoding colored or fluorescent or luminescent proteins (e.g.,green fluorescent protein, enhanced green fluorescent protein, redfluorescent protein, luciferase), and proteins which mediate enhancedcell growth and/or gene amplification (e.g., dihydrofolate reductase).Epitope tags include, for example, one or more copies of FLAG-tag, His,myc, Tap, HA or any detectable amino acid sequence. “Expression tags”include sequences that encode reporters that may be operably linked to adesired gene sequence in order to monitor expression of the gene ofinterest.

Other exemplary cell lines for which expression of the gRNA(s) ornucleic acid editing system constructs may be quantified include: C8161,CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC,HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, C1R, Rath, CV1, RPTE,A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2,P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1,BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRCS, MEF, Hep G2, HeLa B,HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial,BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetalfibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780,A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36,Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr −/−,COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1,CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1,EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-MeI 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, and YAR.

Repair Templates

In certain instances, where a nucleotide substitution, insertion, ordeletion is desired, for example, the compositions, methods, kits, anddelivery systems provided herein further comprise a repair template.Repair templates may comprise any nucleic acid, for example, DNA,messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA),single stranded RNA (ssRNA), or antisense oligonucleotides. In someembodiments, the repair template is a DNA repair template. The basiccomponents and structure of the DNA repair template to support geneediting is known, and described in Ran F A, Genome editing using theCRISPR-Cas9 System, PNAS 8(11):2281-308 (2013); and Pyzocha et al.,RNA-guided genome editing of mammalian cells, Methods Mol. Biol.1114:269-77 (2014) which are hereby incorporated by reference.

The length of the repair template can vary, and can be, for example,from 200 base pairs (bp) to about 5000 bp, such as about 200 bp to about2000 bp, such as about 500 bp to about 1500 bp. In some embodiments, thelength of the DNA repair template is about 200 bp, or is about 500 bp,or is about 800 bp, or is about 1000 base pairs, or is about 1500 basepairs. In other embodiments, the length of the repair template is atleast 200 bp, or is at least 500 bp, or is at least 800 bp, or is atleast 1000 bp, or is at least 1500 bp.

In some embodiments, the repair template is in the same delivery vehicleas the gRNA. In other embodiments, the repair template is in the samedelivery vehicle as the nucleic acid editing system. In someembodiments, the repair template can be present on a contiguouspolynucleotide with the gRNA gene, and the repair template may bedesigned for incorporation by homologous recombination.

In some embodiments, the delivery system provides a gRNA and repairtemplate, wherein the repair template is covalently or non-covalentlybound to the gRNA. In further embodiments, the repair template ispartially annealed to the gRNA. In some embodiments, the repair templateis covalently or non-covalently bound to a gRNA delivered via a viralvector and the nucleic acid editing system comprises a nuclearlocalization signal and is delivered by a non-viral vector, such that itcarries the gRNA along with the repair template to the nucleus of thecell. Thus, in some embodiments, the delivery systems, compositions,methods, and kits disclosed herein greatly improve the percentefficiency of nucleotide sequence modification by providing a system bywhich the repair template is efficiently directed to the nucleus of thecell.

Administration of the Delivery Systems

The delivery vehicles (whether comprising conjugates, viral or non-viralvectors, or a combination thereof) may be administered by any methodknown in the art, including injection, optionally by direct injection totarget tissues. In some embodiments, the gRNA, nucleic acid editingsystem, and, optionally, repair template are administered simultaneouslyin the same or in different delivery vehicles. In other embodiments, thegRNA and nucleic acid editing system and, optionally, repair templateare administered sequentially via separate delivery vehicles. In someembodiments, the gRNA is administered 1, 3, 5, 7, 10, 14, or 30 daysprior to administration of the nucleic acid editing system, such thatthe gRNA accumulates in the target tissue prior to administration of thenucleic acid editing system. In some embodiments, the gRNA and/ornucleic acid editing system is administered in a plurality of doses,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more doses. In various embodiments, the gRNA and/or nucleic acidediting system is administered over a time period of from one week toabout six months, such as from about two to about ten doses within abouttwo months, such as from about three to about five doses over about onemonth.

In one embodiment, the gRNA and, optionally, a repair template, areprovided in an AAV vector that is administered to the subject or cellprior to administration of a nanoparticle containing the nucleic acidediting system. In a further embodiment, the AAV vector comprising thegRNA is administered 3, 4, 5, 6, 7, 8, 9, or 10 days prior to theadministration of the nanoparticle, to allow expression of the gRNA fromthe AAV vector. In a yet further embodiment, the nanoparticle containingthe nucleic acid editing system is administered multiple times, forexample, once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15days. In a still further embodiment, the nanoparticle containing thegRNA is administered for 1 month, 2 months, 3, months, 4 months, 6months, 8 months, 10 months, 12 months, 18 months, 24 months, or longer.Since AAV expression can occur for 2 years or longer, in one embodiment,the expression of the gRNA and, optionally, repair template, from theAAV vector and the continual administration of nanoparticles containingthe nucleic acid editing system provides efficient gene editing of thetarget sequence with reduced or absent off-target effects due to thetransient expression of the nucleic acid editing system.

In another embodiment, the repair template is delivered via an AAVvector, and is injected 3, 4, 5, 6, 7, 8, 9, or 10 days prior to theadministration of nanoparticles containing the nucleic acid editingsystem and/or the gRNA. As described above, the nanoparticles may beadministered multiple times, and for several months. In suchembodiments, the repair template is expressed from the AAV vector in thecell for 2 years or longer, and the nanoparticles comprising the nucleicacid editing system and/or gRNA are administered in multipleadministrations over time in order to provide efficient gene editing ofthe target sequence with reduced or absent off-target effects.

In particular embodiments, one or more gRNA and, optionally, a repairtemplate, is provided in an AAV vector that is administered first, and aCas9 nucleic acid editing system in a lipid-based delivery vehicle issubsequently administered in one or more doses. In some embodiments, theCas9 is administered in a lipid-based delivery vehicle about 7 days andabout 14 days after the administration of the one or more gRNA in an AAVvector.

In another embodiment, each of the components of the delivery systemsprovided herein (e.g., the nucleic acid editing system, gRNA and,optionally, repair template) are each contained in the same or indifferent nanoparticles. In a further embodiment, the nanoparticlescontaining the nucleic acid editing system, gRNA, and, optionally,repair template, are administered at multiple time points, for example,every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In another embodiment,the administration of the nanoparticles separately comprising thenucleic acid editing system and gRNA are administered at different timepoints in order to enhance gene editing efficiency in a particular cellor for a particular disease type.

In some embodiments, the administration of the delivery system iscontrolled so that expression of the nucleic acid editing system istransient. In some embodiments, such transient expression of the nucleicacid editing system minimizes off-target effects, thereby increasing thesafety and efficiency of the gene editing system disclosed herein. Forexample, in expression of the nucleic acid system is controlled viaselection of the delivery vehicles and/or promoters disclosed herein.

In some embodiments, the present disclosure provides compositions andmethods that allow for increased safety and/or efficacy of the nucleicacid editing systems provided herein. For example, in some embodiments,the non-viral delivery of Cas9 results in a reduced or eliminated immuneresponse against Cas9, relative to the immune response elicited againstCas9 when delivered via a viral vehicle. In some embodiments, non-viraldelivery of Cas9 triggers little or no immune response in the subject.For example, the concentration of one or more cytokines (e.g. one ormore of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, IFNγ,G-CSF, and GM-CSF) may, following administration, be about 90%, about95%, about 100%, about 105%, about 110%, or about 120% of the cytokineconcentration compared to a PBS (phosphate-buffered saline) control.Thus, in particular aspects, the concentration range may be about 90% toabout 110%, or about 95% to about 100%, when compared to a PBS control.In some embodiments, the methods provided herein with respect tonon-viral delivery of Cas9 result in temporary Cas9 expression in thecell. Temporary expression of Cas9, in some embodiments, reduces oreliminates off-target effects and/or reduces or eliminates the inductionof immune responses against Cas9 relative to longer-term expression ofCas9 via viral delivery methods.

Advantageously, the non-viral methods disclosed herein provide forrepeated dosing such that the efficiency of gene editing increases witheach dose, in the absence of the immune stimulation and off-targeteffects associated with virally delivered Cas9. For example, in someembodiments, the percent efficiency of gene editing increases by about1%, about 2%, about 5%, about 10%, or more with each subsequent dose.Doses may be from about 0.1 mg/kg to about 300 mg/kg RNA or protein. Forexample, in some embodiments, Cas9 protein or RNA is administered at adose of about 0.1 mg/kg to about 300 mg/kg, or about 0.2 mg/kg to about250 mg/kg, or about 0.3 mg/kg to about 200 mg/kg, or about 0.4 mg/kg toabout 150 mg/kg, or about 0.5 mg/kg to about 100 mg/kg, or about 1 mg/kgto about 50 mg/kg. Doses may be about 1 day apart, about 2 days apart,about 3 days apart, about 4 days apart, about 5 days apart, about 6 daysapart, about 1 week apart, about 2 weeks apart, about 3 weeks apart,about a month apart, or more.

In some aspects, gene editing without any repair template may facilitategene repair. Thus, in some embodiments, the present disclosure providesmethods, compositions, and delivery systems for gene editing whereinnonhomologous end-joining (NHEJ) rather than homology-driven repair(HDR) is achieved. For example, in some embodiments, a loss of functionof a key protein due to an out-of-frame mutation causes a disease ordisorder. For example, in muscular dystrophy, the loss of function of akey protein is due to out of reading frame mutations. Thus, in someembodiments, the present disclosure provides methods, compositions, anddelivery systems wherein a non-functional gene may be restored tofunction via deletion of a fragment of a gene, and wherein the methods,compositions, and delivery systems do not comprise a repair template. Insome embodiments, the present disclosure provides methods, compositions,and delivery systems wherein a splicing defect may be repaired. In otherembodiments, the present disclosure provides methods, compositions, anddelivery systems wherein a gene is deleted, for example, a harmful genethat is associated with or causes a disease or disorder. In someembodiments, NHEJ is achieved at a higher efficiency relative to HDR.For example, in some embodiments, the percent efficiency of NHEJ is fromabout 10% to about 90%.

In another embodiment, the gRNA, Cas9, and, optionally, repair template,are administered to a subject or a cell at the same time, such as on thesame delivery vehicle, and one or more component (i.e., the gRNA, Cas9,and/or repair template) is under the control of an inducible promoter.As an example, in one embodiment, the gRNA, Cas9, and repair templateare each present on an AAV viral vector, and the gRNA is under thecontrol of an inducible promoter, for example, a small molecule-inducedpromoter such as tetracycline-inducible promoter. In a furtherembodiment, the Cas9 is expressed 5-7 days following administration ofthe vector, after which the expression of the gRNA is induced by one ormore injections of the small molecule such as tetracycline. The gRNAexpression can be induced at various time points in order to increasegene editing efficiency; for example gRNA expression may be inducedevery day, or every 2 days, or every 3 days, or every 5 days, or every10 days, or every 2 weeks, for at least 1 week or at least 2 weeks, orat least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least6 weeks, or at least 7 weeks, or at least 8 weeks, or at least 10 weeks,or at least 11 weeks, or at least 12 weeks, or more. Thus, the Cas9expression may be expressed from the AAV vector over time, and the gRNAmay be inducibly expressed by multiple injections of the inducingmolecule over several days, weeks, or months. Similarly, the gRNA can beexpressed from the AAV vector over time, and the Cas9 may be induciblyexpressed under control of an inducible promoter by multiple injectionsof the inducing molecule over several days, weeks, or months. In aparticular embodiment, the AAV vector comprising gRNA on an induciblepromoter, Cas9, and a repair template is administered to the subject orcell on day 1; and a small molecule to induce gRNA expression isadministered to the subject or cell beginning on day 5, 6, 7, or 8, andevery 3 days for several months.

In another embodiment, one or more gRNA and, optionally, a repairtemplate, is delivered via an RNA conjugate, such as an RNA-GalNAcconjugate, and the nucleic acid editing system is delivered via a viralor non-viral vector, such as a nanoparticle. In another embodiment, thegRNA and repair template are attached to the nanoparticle comprising thenucleic acid editing system, such that the components are delivered tothe target cell or tissue together. In such embodiments, the gRNA,repair template, and nucleic acid editing system may be delivered to thetarget cell or tissue together, and expression of each component may becontrolled by way of different promoters, including inducible promoters,as disclosed herein.

In one aspect, the present disclosure provides methods for modifying atarget polynucleotide in a cell, which may be in vivo, ex vivo, or invitro. In some embodiments, the one or more delivery vehicles comprisinga nucleic acid editing system and/or gRNA and, optionally, repairtemplate, are administered to a subject. In further embodiments, thenucleic acid editing system and gRNA and, optionally, repair template,are targeted to one or more target tissues in the subject. For example,in one embodiment, the target tissue is liver, endothelial tissue, lung(including lung epithelium), kidney, fat, or muscle. In one embodiment,the one or more delivery vehicles comprise a viral vector (e.g., AAV) ora non-viral vector such as, for example, MD-1, 7C1, PBAE, C12-200,cKK-E12, or a conjugate such as a cholesterol conjugate or an RNAconjugate as disclosed herein. In one embodiment, the target tissue isliver, and one or more delivery vehicle is MD-1. In another embodiment,the target tissue is endothelial tissue, and one or more deliveryvehicle is 7C1. In another embodiment, the targeting tissue is lung, andone or more delivery vehicle is PBAE or 7C1. In another embodiment, thetarget tissue is kidney, one or more delivery vehicle is an RNAconjugate. In another embodiment, the target tissue is fat, and one ormore delivery vehicle is C12-200. In another embodiment, the targettissue is muscle (e.g., skeletal muscle) and one or more deliveryvehicle is a cholesterol conjugate.

The delivery vehicles (whether viral vector or non-viral vector or RNAconjugate material) may be administered by any method known in the art,including injection, optionally by direct injection to target tissues.Nucleic acid modification can be monitored over time by, for example,periodic biopsy with PCR amplification and/or sequencing of the targetregion from genomic DNA, or by RT-PCR and/or sequencing of the expressedtranscripts. Alternatively, nucleic acid modification can be monitoredby detection of a reporter gene or reporter sequence. Alternatively,nucleic acid modification can be monitored by expression or activity ofa corrected gene product or a therapeutic effect in the subject.

In some embodiments, the subject is a human in need of therapeutic orprophylactic intervention. Alternatively, the subject is an animal,including livestock, poultry, domesticated animal, or laboratory animal.In various embodiments, the subject is a mammal, such as a human, horse,cow, dog, cat, rodent, or pig.

In some embodiments, the methods provided herein include obtaining acell or population of cells from a subject and modifying a targetpolynucleotide in the cell or cells ex vivo, using the delivery systems,compositions, methods, and/or kits disclosed herein. In furtherembodiments, the ex vivo modified cell or cells may be re-introducedinto the subject following ex vivo modification. Thus, the presentdisclosure provides methods for treating a disease or disorder in asubject, comprising obtaining one or more cells from the subject,modifying one or more target nucleotide sequences in the cell ex vivo,and re-introducing of the cell with the modified target nucleotidesequence back into the subject having the disease or disorder. In someembodiments, cells in which nucleotide sequence modification hasoccurred are expanded in vitro prior to reintroduction into the subjecthaving the disease or disorder. In one embodiment, the cells are bonemarrow cells.

In other embodiments, the nucleic acid editing system and gRNA and,optionally, repair template, are administered to a cell in vitro.

In some embodiments, at least one component of the delivery system(e.g., the gRNA or the nucleic acid editing system) accumulates in thetarget tissue, which may be, for example, liver, heart, lung (includingairway epithelial cells), skeletal muscle, CNS (e.g., nerve cells),endothelial cells, blood cells, bone marrow cells, blood cell precursorcells, stem cells, fat cells, or immune cells. Tissue targeting ordistribution can be controlled by selection and design of the viralvector, or in some embodiments is achieved by selection and design oflipid or polymeric particles. In some embodiments, the desired tissuetargeting of the activity is provided by the combination of viral andnon-viral delivery vehicles.

Diseases and Disorders

While the nucleic acid modification system described herein can be usedto make essentially any desired change, in some embodiments the subjecthas a genetic disorder which is sought to be corrected. In some aspects,the disorder is an inborn error of metabolism. In other embodiments, thenucleic acid modification provides a loss of function for a gene that isdeleterious. In some embodiments, the inborn error of metabolism can beselected from disorders of amino acid transport and metabolism, lipid orfatty acid transport and metabolism, carbohydrate transport andmetabolism, and metal transport and metabolism. In some embodiments, thedisorder is hemophilia, cystic fibrosis, or sickle cell disease.Exemplary diseases and conditions that may be treated, prevented oralleviated with the delivery systems, compositions, kits, and methodsprovided herein include: cystic fibrosis, hemophilia, HuntingtonDisease, de Grouchy Syndrome, Lesch-Nyhan Syndrome, galactosemia,Gaucher Disease, CADASIL Disease, Tay-Sachs Disease, Fabry Disease,color blindness, cri du chat, duchenne muscular dystrophy, 22q11.2deletion syndrome, Angelman syndrome, Canavan disease,Charcot-Marie-Tooth disease, down syndrome, Klinefelter syndrome,neurofibromatosis, Prader-Willi syndrome, Tay-Sachs disease,haemochromatosis, phenylketonuria, polycystic kidney disease, sicklecell disease, alpha 1-antitrypsin deficiency (A1AD), and tyrosinemia,growth hormone deficiency, metachromatic leukodystrophy,mucopolysaccharidosis I, phenylketonuria, short chain acyl-CoAdehydrogenase deficiency, alpha-1 antitrypsin deficiency, diabetes,obesity, myocarditis, glomerulonephritis, organophosphate toxicity,xenotransplantation, hypoxic-ischemia encephalopathy, liverregeneration, and various types of cancer, among others.

“Cancer” herein refers to or describes the physiological condition inmammals that is typically characterized by unregulated cell growth. Insome aspects, cancer is a genetic disease or disorder as disclosedherein. Examples of cancer include but are not limited to carcinoma,lymphoma, blastoma, sarcoma (including liposarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, leiomyosarcoma, chordoma,lymphangiosarcoma, lymphangioendotheliosarcoma, rhabdomyosarcoma,fibrosarcoma, myxosarcoma, chondrosarcoma), neuroendocrine tumors,mesothelioma, synovioma, schwanoma, meningioma, adenocarcinoma,melanoma, and leukemia or lymphoid malignancies. More particularexamples of such cancers include squamous cell cancer (e.g. epithelialsquamous cell cancer), lung cancer including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung, small cell lung carcinoma, cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer,tumors of the biliary tract, Ewing's tumor, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, myelodysplastic disease, heavy chain disease,neuroendocrine tumors, Schwanoma, and other carcinomas, as well as headand neck cancer.

Many cancers are characterized by certain gene mutations that promote or“drive” tumorigenesis (“tumor driver genes”), for example, mutations intumor suppressor genes or pro-apoptotic genes. In some embodiments, thepresent disclosure provides methods for treating or preventing cancercomprising use of the compositions, methods, and delivery systemsprovided herein to target tumor driver genes. Advantageously, thecompositions, methods, and delivery systems provided herein allow forrepeated dosing such that a therapeutic effect can be achieved. Tumordriver genes, oncogenes, and tumor suppressors are known in the art andinclude, but are not limited to, APC, beta-catenin, CYLD, HIN-1, KRAS2b,p16, p19, p21, p2′7, p27mt, p53, p5′7, p′73, PTEN, MDA-7, Rb,Uteroglobin, Skp2, BRCA-1, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18,mda7, MEN1, MEN2, MTS1, NF1, NF2, VHL, WRN, WT1, CFTR, C-CAM, CTS-1,zacl, ras, MMAC1, FCC, MCC, FUS1, Gene 26 (CACNA2D2), Yap gene, PL6,Beta (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, andGene 21 (NPRL2). Pro-apoptotic genes are known in the art and include,but are not limited to, CD95, caspase-3, Bax, Bag-4, CRADD, TSSC3, bax,hid, Bak, MKP-7, PARD, bad, bcl-2, MST1, bbc3, Sax, BIK, and BID. Thepresent disclosure also provides for the treatment or prevention ofcancer by targeting tumor driver genes as well as targeting immunesuppressor genes associated with cancer. Immune suppressors associatedwith cancer are known in the art and include, for example, IDO, FasL,VEGF, IL-10 TGF-β, TRAIL DAF, i NOS, CTLA-4, and STAT3.

In some embodiments, a tumor suppressor, oncogene and/or immunesuppressor gene is targeted, and a paracrine effect is achieved via themethods, compositions, and delivery systems provided herein. Forexample, in some embodiments, tumor suppressing effects can be increasedor optimized even where the gene editing is not 100% efficient, becausethe targeted tumor cells or cells in the tumor microenvironment willallow further tumor suppressing effect or activate the immune responseagainst the tumor for further tumor suppressing effect.

Exemplary genetic disorders that can be treated or ameliorated invarious embodiments, as well as target genes that can be edited forimproved or reduced activity, are disclosed in U.S. Pat. No. 8,697,359,which are hereby incorporated by reference.

Therapeutic Effect

“Therapeutic effect” as used herein refers to an effect on a disease orcondition that is a measurable improvement in the progression, symptoms,or phenotype of the disease or condition. A “therapeutic treatment” or“therapeutically effective amount” provides a therapeutic effect in asubject. A therapeutic effect may be a partial improvement or may be acomplete resolution of the disease or disorder. A therapeutic effect mayalso be an effect on a disease or condition as measured using a testsystem recognized in the art for the particular disease or condition. Atherapeutic effect may also be a prophylactic effect, such that thedisease or condition may be prevented, or such that symptoms of anunderlying disease or condition may be prevented before they occur. Forexample, the delivery systems, methods, compositions, and kits disclosedherein may be used to correct or improve a gene product such that theonset of a disease or condition, or an infection with an infectiousagent, is prevented. A “gene product” as used herein refers to a productof gene expression. In various embodiments, the gene product is aprotein or enzyme; however, a gene product may also be RNA (e.g., whenthe gene codes for a non-protein product such as functional RNA).

In one aspect, the delivery systems, compositions, methods, and kitsdisclosed herein are useful for therapeutic treatment of geneticdiseases and disorders, cancers, immune system disorders, or infectiousdiseases. In some embodiments, the diseases, disorders, and cancers areassociated with mutations that cause expression of one or more defectivegene products, or cause an aberrant increase or decrease in theproduction of a gene product. In some embodiments, the therapeuticefficacy of the delivery systems, compositions, methods, and kitsdisclosed herein may be assessed or measured by expression level oractivity level of the product of the targeted nucleotide sequence. Insome embodiments, gene loci are sequenced by Sanger or Next GenerationSequencing. In some embodiments, in human subjects or other subjects, atherapeutic effect or the therapeutic efficiency of the compositions andmethods for target sequence modification disclosed herein may bemeasured or monitored using surrogate markers of efficiency. Surrogatemarkers of efficiency may be, for example, an improvement in a symptomof the disease or condition; a clinical marker such as, for example,liver function; expression of a wild-type gene product or an improvedgene product relative to the gene product that was expressed in the cellor subject prior to treatment; expression of a sufficient amount oractivity of the gene product to improve or resolve the disease ordisorder; or expression of the gene product in a manner that providesany other therapeutic effect. In some embodiments, surrogate markers mayinclude serum markers such as, for example, factor VIII and/or IX. Forexample, factor VIII and factor IX can be measured as surrogate markersfor efficiency of treatment for hemophilia A and hemophilia B,respectively. In some embodiments, any gene product excreted throughexosome to the serum can be detected by purification and sequencing.

In some embodiments, the disease or disorder may be therapeuticallytreated using the methods, compositions, kits, and delivery systemsdisclosed herein, wherein an efficiency rate of target sequencemodification or an efficiency rate of gene product modification is atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90% at least 95%, or atleast 99%. In some embodiments, the disease or disorder may betherapeutically treated using the methods, compositions, kits, anddelivery systems disclosed herein, wherein an efficiency rate of targetsequence modification or an efficiency rate of gene product modificationis less than 100%, or wherein an effect on fewer than 100% of the cellsin the relevant tissue, has a therapeutic effect in the subject. Forexample, a therapeutic effect may be achieved when the percentefficiency of nucleic acid modification may be about 0.01% to about100%, about 0.01% to about 50%, about 0.05% to about 40%, about 0.1% toabout 30%, about 0.5% to about 25%, about 1% to about 20%, about 1% toabout 15%, about 1% to about 10%, or about 1% to about 5%. Thus, even ifthe efficiency of nucleotide sequence modification is relatively low(e.g., less than 50%, or less than 40%, or less than 30%, or less than20%, or less than 10%, or less than 5%, or less than 1%, or less than0.5%, or less than 0.1%), modest expression of the introduced orcorrected or modified gene product may result in a therapeutic effect inthe disease or disorder.

Thus, in some embodiments, a genetic disease or condition may beimproved or resolved even if the target nucleotide sequence is onlymodified in a fraction of the target population of cells in the subject.In some embodiments a percent efficiency of nucleic acid modification ofless than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, less than 1%, less than 0.5%, or less than 0.1%nevertheless results in high level expression of an introduced orcorrected gene and thereby resolves the genetic disorder by providingsufficient expression of the relevant product.

In some genetic disorders, the presence of only a few improved orcorrected gene product results in a measurable improvement or evenresolution of the disease or disorder. For example, without wishing tobe bound by theory, disorders in which disease is caused by a simpledeficiency of a particular gene product may be resolved with a limitednumber of nucleotide sequence modifications. For example, recessivelyinherited disorders are often simple loss-of-function mutations, andoften there is a wide variation in the normal levels of gene expression(e.g., heterozygotes often have about 50% of the normal gene product andare asymptomatic), such that expression of a relatively small percentageof the normal gene product may be sufficient to resolve the disorder. Onthe other hand, dominantly inherited disorders in which heterozygotesexhibit loss-of-function with 50% of the normal gene product may, insome embodiments, require a higher level of nucleotide sequencemodification in order to achieve a therapeutic effect. For example, andwithout wishing to be bound by theory, disorders such as cystic fibrosisan Muscular dystrophy (MD) may exhibit a therapeutic effect upon anefficiency of about 1% to about 40%; hemophilia A and B, galactosemia,primary hyperoxaluria, hepatoerythropoietic porphyria, and Wilson'sdisease may each exhibit a therapeutic effect upon achieving anefficiency of about 1% to about 5%; and alpha 1-antitrypsin deficiency,hereditary tyrosinemia type I, Fanconi's anemia, and junctionalepidermolysis bullosa may each exhibit a therapeutic effect uponachieving an efficiency of about 0.1% to about 5%. A percent efficiencyof nucleic acid modification may be directly measured in animal modelsor in in vitro assays by measuring the percent of cells in the targetpopulation in which the target nucleotide sequence has been modified.Or, a percent efficiency of nucleic acid modification may be indirectlymeasured, such as by using surrogate markers as described above.

In some embodiments, the method modifies a target sequence that is agenetic variant selected from a single-nucleotide polymorphism (SNP),substitution, insertion, deletion, transition, inversion, translocation,nonsense, missense, and frame shift mutation. In other embodiments, thetarget sequence is a sequence from an infectious agent, such as a virusor provirus. A provirus is a viral genome that has integrated into theDNA of a host cell. Proviruses may be retroviruses or other types ofviruses that are capable of integration into a host genome. For example,adeno-associated viruses (AAV) have been shown to be capable of hostchromosome integration. Other proviruses include, without limitation,HIV and HTLV.

In some embodiments, the delivery systems and compositions disclosedherein are formulated such that the ratio of the components is optimizedfor consistent delivery to the target sequence and/or consistentresolution of the disease or disorder. In one embodiment, the ratio ofthe gRNA and nucleic acid editing system is optimized for consistentdelivery to the target sequence and/or consistent resolution of thedisease or disorder. In another embodiment, the ratio of the repairtemplate to the gRNA and/or to the nucleic acid editing system isoptimized for consistent delivery to the target sequence and/orconsistent resolution of the disease or disorder. For example, in someembodiments, the delivery systems provide expression of an optimalnumber of gRNAs such that upon delivery to the cell, target tissue, orsubject, the modification of target nucleotide sequences by the gRNA andnucleic acid editing system and, optionally, repair sequence, can bemaximized. For example, in one embodiment, the ratio ofCas9:sgRNA:template is from about 1:1:1 to about 1:1:100. In a furtherembodiment, the ratio is from about 1:1:2 to about 1:1:90, from about1:1:5 to about 1:1:75, or from about 1:1:10 to about 1:1:50. In otherembodiments, the ratio is about 1:1:1 or below, such as from about1:1:0.01 to about 1:1:1, from about 1:1:0.02 to about 1:1:0.75, or about1:1:0.05 to about 1:1:0.5, or about 1:1:0.1 to about 1:1:0.5. Forexample, in some embodiments, the ratio of Cas9:sgRNA:template is 1:1:1or below when NHEJ is inhibited or when Cas9 is fused with one or moreproteins that can facilitate HDR. In other embodiments, wherein thedelivery systems do not comprise a repair sequence, the ratio ofCas9:sgRNA is from about 1:100 to about 100:1, or about 1:50 to about50:1, or about 1:25 to about 25:1, or about 1:10 to about 10:1, or about1:5 to about 5:1, or about 1:2 to about 2:1, or about 1:1.

In one aspect, the present disclosure provides methods for safe andefficient delivery of a nucleic acid editing system via a non-viralvector delivery system, or via a system that includes a viral vector aswell as a non-viral vector, such that off-target effects (e.g.,off-target effects due to long term expression of a nucleic acid editingsystem and a gRNA through genome integration of an AAV vector deliverysystem) are minimized. In some embodiments, the present disclosureprovides methods for delivery of a nucleic acid editing system thatprovide a favorable safety margin. By “favorable safety margin,” ismeant that the compositions and methods provided herein provide geneediting that is both safe and efficient according to the efficiencydeterminations provided herein. Safety may be determined by any methodknown in the art, for example, low off-target effects and/or minimalcytotoxicity and/or intact or normal organ histology (e.g., liverhistology), and/or normal serum biochemistry and/or normal levels ofserum cytokines. Safety may further be determined by comparing theoff-target effects and/or cytoxicity and/or organ histology and/orbiochemistry and/or serum cytokines with other methods of gene editingknown in the art. In one aspect, the present disclosure provides a safeand efficient method for gene editing comprising administering to a cellor subject a Cas9 nucleic acid editing system in a lipid-based vector,e.g., a vector comprising C12-200, cholesterol, C14-PEG 2000, DSPC andCas9 mRNA. In some embodiments, the Cas9 mRNA encodes the Cas9 proteinwith chemical modifications to decrease immune stimulation, as providedherein.

In one aspect, the disclosure provides kits containing any one or moreof the components disclosed in the above methods, compositions, anddelivery systems. Kit components may be provided individually or incombinations, and may be provided in any suitable container, such as avial, a bottle, or a tube. In some embodiments, the kits disclosedherein comprise one or more reagents for use in the embodimentsdisclosed herein. For example, a kit may provide one or more reaction orstorage buffers. Reagents may be provided in a form that is usable in aparticular assay, or in a form that requires addition of one or moreother components before use (e.g. in concentrate or lyophilized form).Suitable buffers include, but are not limited to, phosphate bufferedsaline, sodium carbonate buffer, sodium bicarbonate buffer, boratebuffer, Tris buffer, MOPS buffer, HEPES buffer, and combinationsthereof. In some embodiments, the buffer is alkaline. In someembodiments, the buffer has a pH from about 7 to about 10.

For example, a kit may comprise: (1) a gRNA and (2) a nucleic acidediting system. The kit may further comprise a repair template. The kitmay provide (1) an expression system providing for expression of a gRNAin a target cell or target tissue for at least 2 weeks, the gRNAdirecting cleavage of a target nucleic acid sequence in the targettissue by a nucleic acid editing system, and the expression systemoptionally comprising a repair template, and (2) one or more doses of anRNA delivery system, each dose providing for expression of the nucleicacid editing system in the target tissue for no more than about onemonth. In various embodiments, the kit may provide from two to ten dosesof the RNA delivery system, which may be administered over a time periodof from one week to about two months. In some embodiments, the kitcontains from about two to about five unit doses.

The kit may be custom made to repair a genetic disorder, such as aninborn error of metabolism, or a cancer. In other embodiments, thenucleic acid modification provides a loss of function for a gene that isdeleterious. In some embodiments, the inborn error of metabolism can beselected from disorders of amino acid transport and metabolism, lipid orfatty acid transport and metabolism, carbohydrate transport andmetabolism, and metal transport and metabolism. In some embodiments, thedisorder is hemophilia, cystic fibrosis, or sickle cell disease.

EXAMPLES Example 1 Correction of Gene Defect by Two Delivery Vehicles

The type II bacterial clustered, regularly interspaced, palindromicrepeats (CRISPR)-associated (Cas) system has been engineered into apowerful genome editing tool consisting of the Cas9 nuclease and asingle gRNA (sgRNA). The sgRNA targets Cas-9 to genomic regions that arecomplementary to the 20-nucleotide target region of the sgRNA and thatcontain a 5′-NGG-3′ protospacer-adjacent motif (PAM). Double-strandedDNA breaks generated by Cas9 at target loci are repaired bynon-homologous end-joining or homology-directed repair (HDR). We havedemonstrated CRISPR-Cas9-mediated correction of a Fumarylacetoacetatehydrolase (Fah) mutation in hepatocytes in a mouse model of the humandisease hereditary tyrosinemia. Delivery of components of theCRISPR-Cas9 system by hydrodynamic injection resulted in initialexpression of the wild-type FAH protein in ˜1/250 liver cells. Expansionof FAH-positive hepatocytes rescued the body weight loss phenotype.Example 1 demonstrates the use of a viral and non-viral vector deliveryvehicles administered sequentially in vivo, along with repair templateto correct a FAH gene mutation in the liver. Specifically, using theCRISPR-Cas9 system and combining a lipid nanoparticle for mRNA deliveryand a viral vector for DNA delivery, FAH genes were corrected in theliver of adult mice.

Specifically, an AAV-2/8 virus was designed to express a gRNA and toprovide a DNA repair template. Cas9 mRNA was encapsulated in lipidnanoparticles using cKK-E12, DOPE, Cholesterol, and C14_PEG2000. Thislipid nanoparticle showed significant delivery of Cas9 mRNA to liver.Injecting AAV-2/8 virus to express a gRNA against the mutant FAH geneand a correct FAH repair template, and lipid nanoparticles encapsulatedCas9 mRNA, provided efficient in vivo gene correction.

FIG. 1 shows the design of DNA carried by Adeno-Associated Virus (AVV)2/8, which can deliver DNA to liver of human and rodents with highefficiency. After packing the designed DNA sequence into the AAV 2/8,the vector can express through a U6 promoter a sgRNA targeting theregion of the FAH mutation (Exon 8 of FAH). Meanwhile, a repair templatewas provided in the same vector. The repair template contains thecorrect sequence harboring a “G” rather than “A” at the mutated site.The left and right arm is about 800 bp each. Because AAV is a singlestand DNA virus, the AAV with such sequence can be used as a repairtemplate. The FM and R10 primer can be used to amplify the region forfurther analysis.

FIGS. 2A-B shows packing Cas9 mRNA into cKK-E12. (A) The structure oflipid nanoparticle carrying Cas9 mRNA. cKK-E12 is a house developedlipid. Cas9 mRNA is purchased from TriLink BioTechnologies. Cas9 mRNAexpresses a version of the Streptococcus pyogenes SF370 Cas9 protein(CRISPR Associated Protein 9) that has been codon optimized forexpression in mammalian systems and contains a C-terminal nuclearlocalization signal followed by a human influenza hemagglutinin (HA)tag. This capped and polyadenylation mRNA is optimized for mammaliansystems and modified to reduce immune stimulation. It mimics a fullyprocessed mature mRNA. (B) The lipid nanoparticle was injected to FVB/Nmice at 1 mg/kg, and liver harvested 24 hours later. Western blot wasperformed to detect HA tag in Cas9. The Cas9 protein is about 140-150 kdin protein gels, and was detected by HA antibody at 1:1000 dilution.

FIGS. 3A-B shows analysis of FAH mutant mice that received the AAV 2/8delivery vehicle on Day 0, and at Day 7 and Day 14, received 1 mg/kg ofthe lipid nanoparticle encapsulated Cas9 mRNA (or PBS as a control). AtDay 21, mice were sacrificed and liver taken for immunohistochemistrystaining using FAH antibody. (A) Low resolution picture to show a largearea of liver. (B) High resolution.

In conclusion, efficient in vivo gene editing by combination of viraland non-viral vector to deliver CRISPR-Cas system and a template forediting.

Example 2 In Vitro Testing System

An in vitro testing system for the gene editing system described inExample 1 was established. An sgRNA targeting EGFP was expressed inHEK293T cells, which overexpress EGFP. Cas9 mRNA-LNP was added to thecells in vitro and EGFP expression was assessed by flow cytometry. Theresults of the study are provided in FIG. 4. Untreated HEK293T cellsexpressing the EGFP-targeting sgRNA expressed high levels of EGFP (leftpanel). When MD-1-Cas9mRNA or C12-200-Cas9mRNA were added to the HEK293Tcells (center panel or right panel, respectively), EGFP expression wasknocked down in over 45% of the cells.

Thus, the study indicated that in vitro delivery of Cas9 via a non-viralvector such as a lipid nanoparticle to cells expressing a sgRNA resultsin robust reduction of expression of the nucleic acid targeted by thesgRNA.

Example 3

Studies were conducted to optimize the sgRNAs in the FAH mouse modelsystem. Mutations were detected using a Surveyor assay, and the resultsof the studies are provided in FIGS. 5-7. The surveyor assay is a robustmethod to detect mutations and polymorphisms in DNA. Suveryor nucleaserecognizes and cleaves all types of mismatches arising from the presenceof single nucleotide polymorphisms or from small insertions ordeletions. Surveyor assay is based on the generation of PCR productsthat are subsequently hybridized to generate mismatches inheteroduplexed DNA, which is then treated and cleaved by Surveyornuclease. Size based fragmentation analysis is performed to detectcleaved DNA.

FIG. 5 provides the selection of the most potent sgRNA in an in vitrosystem. In this system, mouse embryonic fibroblasts were isolated fromFAH mice and immobilized by shRNA P53; transfection efficiency wasincreased by overexpression of mutated Ras gene. The system reached morethan 90% transfection efficiency. FIG. 6 provides the selection of themost potent sgRNA in vivo. For the in vivo assay, FAH mice wereadministered sgRNA via hydrodynamic injection, and a surveyor assay wasperformed to determine the efficiency of indels formation. The in vitroand in vivo correlations of indels formation obtained from theseexperiments are provided in FIG. 7. The results of the study showed thatFAH23 provided the best efficiency both in vitro and in vivo.

Example 4

Lipid Nanoparticle-Mediated Delivery of Cas9 mRNA in Liver DiseaseTherapy

A non-viral delivery of Cas9 mRNA allows for a shorter tem expressionand eventual removal of the nuclease from the body. A systemic deliveryof Cas9 mRNA by lipid nanoparticles and sgRNA/HDR template by AAV wasperformed through a method of treating Fah^(mut/mut) mice.

Cas9 mRNA was formulated with C12-200, which is a lipid-like materialutilized in facilitating siRNA delivery in rodents and primates. Cas9mRNA was also formulated with associated helper lipids. Nanoparticleswere formulated with Cas9 mRNA, which was chemically modified to reduceTLR responses,

Mice Study

All animal experiments were performed under the guideline of the MITAnimal Care and Use Committee. Fah^(mut/mut) mice were kept on 10 mg/LNTBC water. Mice with more than 20% weight loss were humanely euthanizedaccording to MIT protocol. 1 or 2 mg/kg nano.Cas9 mRNA and 6e11 genomecopy AAV8 were injected into 9-11 weeks old Fah^(mut/mut) mice throughtail vein. To measure initial repair rate, Fah^(mut/mut) mice were kepton NTBC water.

Cas9 mRNA Nanoparticle Formulation

Cas9 mRNA encodes the Cas9 protein with chemical modification ofpseudouridine and 5-methylcytidine to decrease immune stimulation(Trilinkbiotech). Nano.Cas9 was formulated with C12-200, cholesterol,C14-PEG 2000, DSPC and Cas9 mRNA in a weight ratio of 50:20:10:10 usingmicrofluidic method.

Construction of AAV Vectors and Virus Production

AAV vector was constructed using Gibson assembling. AAV2/8 virus wereprepared and purified by vector cores at Boston Children's HospitalViral Core.

Liver Histology, Serum Markers, and Cytokines

Mice were humanely sacrificed by CO2. Livers were freshly fixed with 4%PFA (paraformaldehyde) and embedded in paraffin. 4 μm sections werestained with hematoxylin and eosin (H&E) for pathology and with anti-Fah(Abcam, 1:400) antibody for immunohistochemistry, as described in, forexample, Xue et al. Response and resistance to NF-kappaB inhibitors inmouse models of lung adenocarcinoma. Cancer Discov. 1:236-247 (2011).The percentage of positive cells was measured at low magnification lensfrom >3 regions per liver in at least 3 mice per group. Blood wascollected using retro-orbital puncture at terminal time point. ALT, ASTand bilirubin levels in serum were measured as described in, forexample, Yin et al. Genome editing with Cas9 in adult mice corrects adisease mutation and phenotype. Nat. Biotechnol. 32:551-553 (2014).Cytokine levels in plasma were determined by Multi-Analyte ELISArray(Qiagen).

Gene Expression Analysis and qRT-PCR

RNA was purified using Trizol (Invitrogen) and reverse-transcribed usinga High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Real-time PCR (qPCR) reactions were performed using gene specificprimers (Roche 480). Data were normalized to Actin.

Cell Culture, Off-Target Analysis, Illumina Sequencing, and Statistics

293T cells were infected with lentivirus to stably express EF1a-GFP(addgene 26777) and U6-sgGFP, as described in, for example, Gilbert etal. CRISPR-mediated modular RNA-guided regulation of transcription ineukaryotes. Cell. 154:442-451 (2013). Cells were incubated withnano.Cas9 mRNA. GFP+ cells were counted by FACS. Off-target sitesprediction was using http://crispr.mit.edu/. Deep sequencing librarieswere prepared from ˜1 ng purified PCR products using Nextera XT kits(Illumina). Libraries at equal molar ratio were sequenced on IlluminaNextSeq500 (75 bp, paired-end) or MiSeq machines (150 bp, paired-end) orSingle molecule labelled. Reads were mapped to reference sequences usingbwa with custom scripts. P-values were determined by Student's t-testsand One-Way ANOVA with Tukey post-test using Prism 5 (GraphPad).

Results

To explore whether lipid nanoparticles can deliver Cas9 (Streptococcuspyogenes Cas9) mRNA, we examined the potential of formulated mRNA todeliver Cas9 mRNA formulated with C12-200. Nanoparticles were formulatedwith Cas9 mRNA chemically modified to reduce TLR responses usingcontrolled microfluidic mixing systems (FIG. 11A). These particles(termed nano.Cas9 hereafter) appear in spherical in morphology with atextured interior under Cryo-TEM (FIG. 11B). The mean particle diameterof nano.Cas9 is about 120 nm as determined by dynamic light scattering(FIG. 11C). The particle size of nano.Cas9 was the same on day 0, 7, 11and 18 (FIGS. 11D and 11E), indicating these particles are stable for atleast 18 days in PBS solution. To test whether nano.Cas9 was functional,we used a 293T reporter cell line stably expressing a GFP reporter and aGFP targeting sgRNA (sgGFP) (FIG. 8A). Cas9-mediated frameshiftnonhomologous end-joining (NHEJ) events will result in GFP-negativecells. 293T cells were incubated with 0.4μg/m1 nano.Cas9 and GFP signalwas measured by FACS at 5 days. As shown in FIG. 8B, 77.1±2.6% of cells(n=3) became GFP negative after nano_Cas9 treatment, suggesting thatnanoparticle delivery of Cas9 mRNA can mediate genome editing in cells.To confirm that the GFP negative cells were caused by Cas9 editing, weperformed deep sequencing of the GFP provirus region from genomic DNA(n=2). We observed insertional or deletional mutations (indels)surrounding the Cas9 cleavage site (FIGS. 8D-E). Most indels areframeshift (eg, lnt and 2nt) mutations which cause loss-of-function ofthe GFP reporter. These data suggest that lipid nanoparticles caneffectively deliver Cas9 mRNA in cultured cells.

While lipid-nanoparticle delivery of siRNA to liver in vivo has beenreported, the systemic delivery of mRNA has only recently beendeveloped. To determine whether C12-200 lipid nanoparticles cansystemically deliver Cas9 mRNA to adult animals, we intravenous (i.v.)injected C12-200 lipid nanoparticles encapsulated β-galactosidase(β-gal) mRNA or Cas9 mRNA (FIG. 12A). The size of β-gal mRNA (3.3 kb) isclose to Cas9 mRNA (4.5 kb), and the activity of β-gal protein can bedetected by enzyme reaction. β-gal protein is detected in mouse liverusing immunoblot at 14 hrs after a single dose administration (1 mg/kgor 2 mg/kg), and the amount of protein expressed correlated with thedose of mRNA (FIG. 12B). To investigate whether β-gal is functional invivo, we detected its enzyme activity in mouse liver. Majority of thecells in liver sections stained positive in β-gal activity assay (FIG.12C), suggesting systemic delivery of long mRNA can produce functionalprotein within most of the cells in mouse liver. To determine whetherlipid nanoparticles can deliver Cas9 mRNA, nano.Cas9 (1 mg/kg or 2mg/kg) was injected i.v, and Cas9 protein in total liver lysates wasdetected by immunoblot (FIG. 12D). To measure the half-life of Cas9 mRNAin vivo, total RNA of liver is extracted and qPCR performed. The Cas9mRNA presented in liver at 4 hrs and 14 hrs but was significantlydiminished at 24 hrs, consistent with transient expression.

To determine the safety of nano.Cas9, we first compared the cytotoxicityof nano.Cas9 with Lipofectamine 2000 in 293T cells. Significant toxicitywas observed at the dose of more than 1 μg/ml mRNA-Lipofectamine 2000complex, in contrast, nano.Cas9 was well tolerated at 4 μg/ml (FIG.13A). In contrast, high efficient gene editing (77.1±2.6%) can bereached at the dose of 400 ng/ml nano.Cas9 at 293T cells (FIG. 8),suggesting its favorable safety margin. The nano.Cas9 (2 mg/kg) is welltolerated in animals, as indicated by intact liver histology, normalserum biochemistry and cytokine levels in plasma (FIGS. 13B-D).

To investigate whether nano.Cas9 can be applied for genome editing invivo, we used the Fah^(mut/mut) mouse model of HTI. These mice possessthe same G->A mutation in exon 8 as the common form of this humandisease. To enable repair of the Fah gene, we designed an AAV vectorwith a U6-sgRNA expression cassette and an HDR template (termed AAV-HDRthereafter), which consists of 1.7kb homologous sequence to the Fahgenomic region (FIG. 9A). We designed the HDR template to perform twotasks (1) “G” to repair the mutant “A” (2) “CC” to mutate the PAMsequence to prevent self-cleavage (FIG. 9A). These were packaged usingan AAV2/8 serotype, which has shown the ability to target the liver. Toexplore whether the nano.Cas9 and AAV-HDR combination treatment canrepair the Fah mutation in vivo, a cohort of Fah^(mut/mut) mice (n=3)were i.v. injected with 6e1 1 genome copies of AAV-HDR (FIG. 9B) at Day−14, 2 mg/kg nano.Cas9 at Day −7 and taken off NTBC water at Day 0 (FIG.9B). Mice treated with PBS, AAV-HDR alone or nano.Cas9 alone serve ascontrols. As shown in FIG. 9C, nano.Cas9+AAV-HDR completely preventedbody weight loss upon NTBC water withdrawal, whereas control micerapidly lost 20% body weight and had to be sacrificed. All the mice innano.Cas9+AAV-HDR group survived after 30 days post NTBC withdrawal. At30 days after NTBC water withdrawal, liver histology and serumbiomarkers (AST, ALT) indicated that liver damage was rescued in nanoCas9+AAV-HDR treated mice compared to control mice (FIGS. 9D, 9E).Immunohistochemistry staining also detected patches of Fah positivehepatocytes (FIG. 9F), representing a fraction of total hepatocytes.

To determine the initial Fah gene repair rate in vivo, we injectedFah^(mut/mut) mice with nano.Cas9 and AAV-HDR and kept the mice on NTBCwater to prevent expansion of Fah corrected cells (FIG. 10A). As shownin FIGS. 10B and 10C, 4.6% hepatocytes stained positive of Fah proteinby immunohistochemistry in nano.Cas9 plus AAV-HDR treated animals. Thenumber of Fah positive hepatocytes is correlated with the dose ofnano.Cas9 (FIG. 10C). To investigate whether Fah splicing is restored inthe liver, we performed qRT-PCR using primers spanning Fah exons 8 and 9and observed 9.5% FAH mRNA expression was restored without selection(FIG. 10D). Sanger sequencing of the RT-PCR bands in nano.Cas9+AAV-HDRtreated mice confirmed that the corrected G nucleotide is presented atthe end of exon 8. To examine genome editing in the liver, we performeddeep sequencing of the Fah locus in liver genomic DNA. We observed anaverage of 11.1% indels at predict sgRNA target region within nano.Cas9(2 mg.kg)+AAV-HDR group (n=3 mice).

CRISPR/Cas9 may cause indels at off-target genomic sites, and in orderto determine potential off-target effects after Cas9 mRNA delivery invivo, we performed deep sequencing at three of the top ranking predictedoff-target sites. Compared to indels at the on-target Fah site, indelswere detected at the assayed off-target sites in nano_Cas9+AAV-HDRtreated mouse and these numbers are comparable with AAV alone treatedmouse, suggesting that Cas9 mRNA delivery has low off-target effects atassayed sites.

Therapeutic editing has broad potential to treat a range of diseasesthrough the permanent correction of genetic defects. Through combiningviral and nonviral nucleic acid delivery we report the firsttherapeutically relevant formulations capable of inducing repair of adisease gene in an adult animal, and further advancing the technology ofgene editing. Herein we reported that therapeutic delivery ofCRISPR/Cas9 using mRNA and AAV combination can effectively correct theFah mutation and cure a mouse model of tyrosinemia. Systemic delivery ofCas9 mRNA by lipid nanoparticle and sgRNA/HDR template by AAV correctedFah mutation and restored Fah splicing in more than 1/25 hepatocytes inadult mouse liver. This treatment is well-tolerated in mice and fullyrescued body weight loss and liver damage in tyrosinemia mice.

We showed that systemic delivery of Cas9 mRNA by lipid nanoparticle caneffectively mediate genome editing in vivo. This transient Cas9 mRNAdelivery method provides a platform for non-viral CRISPR/Cas9 delivery.Administration of Cas9 mRNA using non-viral and transient expressionvehicles can allow repeated dosing to increase efficiency and canpotentially prevent long-term side-effects, such as potentialimmune-response against Cas9 and off-target editing. Our mRNA deliverymethod is amenable to deliver Cas9 nickase to reduce off-targetingeffects or therapeutic mRNA such as Fah or Erythropoietin.

We applied AAV, a well-studied clinical viral vehicle, to deliver sgRNAand HDR template to the liver. Because AAV serotypes target a wide rangeof tissue in vivo, our method can target organs other than liver throughengineering of mRNA delivery tools. This study has demonstrated thattherapeutic delivery of Cas9 mRNA and AAV can correct genetic mutationin mice.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

1-160. (canceled)
 161. A method for inducing repair of a gene in asubject in need thereof, the method comprising administering to thesubject a delivery system comprising (i) one or more guide RNA (gRNA)and (ii) a nucleic acid editing system, wherein the one or more gRNA isprovided in a first delivery vehicle and the nucleic acid editing systemis provided in a second delivery vehicle; wherein the first deliveryvehicle and the second delivery vehicle are separate delivery vehicles.162. The method of claim 161, wherein the first delivery vehicle is aviral vector.
 163. The method of claim 162, wherein the viral vector isselected from the group consisting of adeno-associated virus (AAV),adenovirus, retrovirus, and lentivirus vectors.
 164. The method of claim161, wherein the second delivery vehicle is a non-viral vector.
 165. Themethod of claim 164, wherein the non-viral vector is a lipid-based orpolymeric vector.
 166. The method of claim 165, wherein the lipid-basedor polymeric vector is selected from the group consisting of lipids,liposomes, lipid encapsulation systems, nanoparticles, small nucleicacid-lipid particle (SNALP) formulations, polymers, and polymersomes.167. The method of claim 161, wherein the nucleic acid editing system isa CRISPR-Cas system.
 168. The method of claim 161, wherein the deliverysystem further comprises a repair template.
 169. The method of claim168, wherein the repair template is selected from the group consistingof a DNA repair template, an mRNA repair template, an siRNA repairtemplate, an miRNA repair template, and an antisense oligonucleotiderepair template.
 170. The method of claim 168, wherein the repairtemplate is provided in the first delivery vehicle.
 171. The method ofclaim 161, wherein the gRNA hybridizes to a target sequence in a cell inthe subject.
 172. The method of claim 171, wherein the target sequenceis associated with a genetic disease or disorder or a cancer.
 173. Themethod of claim 172, wherein the genetic disease or disorder is aninborn error of metabolism selected from disorders of amino acidtransport and metabolism, lipid or fatty acid transport and metabolism,carbohydrate transport and metabolism, and metal transport andmetabolism.
 174. The method of claim 172, wherein the genetic disease ordisorder is hemophilia, cystic fibrosis, or sickle cell disease. 175.The method of claim 161, wherein the subject is an animal.
 176. Themethod of claim 161, wherein the delivery vehicle comprising the gRNA isadministered prior to the delivery vehicle comprising the nucleic acidediting system.
 177. The method of claim 161, wherein the methodachieves a modification rate of about 1% to about 10% of a population ofcells in a target tissue.
 178. A method for modifying a targetnucleotide sequence in a subject in need thereof, comprising:administering a first delivery vehicle comprising a guide RNA (gRNA),wherein the gRNA is expressed in a target tissue for at least 2 weeks;and administering a second delivery vehicle comprising a CRISPR-Cassystem comprising a Cas mRNA, wherein the Cas mRNA is expressed in thetarget tissue for no more than about two months per administration,wherein the gRNA directs cleavage of a target nucleic acid sequence inthe target tissue by the CRISPR-Cas system.
 179. The method of claim178, wherein the first delivery vehicle comprises a DNA repair template.180. The method of claim 178, wherein the first delivery vehicle is aviral vector.